Biologically pure culture of actinoplanes capable of producing antibiotic A/16686

ABSTRACT

The present invention relates to a glycopeptide antibiotic arbitrarily designated as A/16686 antibiotic, and to its physiologically-acceptable acid addition salts. Antibiotic A/16686 free base, and its acid addition salts are antibacterial agents, especially active against grampositive microorganisms. The process for producing antibiotic A/16686 by culturing a novel strain of the actinoplanes genus, designated as Actinoplanes sp. ATCC 33076, is a further object of the present invention.

This is a division of application Ser. No. 135,560, filed Mar. 31, 1980now U.S. Pat. No. 4,303,646 Dec. 1, 1981.

This invention relates to an antibiotic substance arbitrarily designatedherein as A/16686 antibiotic, to a process for producing it by culturinga hitherto underscribed strain which has been characterizedtaxonomically as a novel strain of the Actinoplanes genus, and to itsuse as an antibacterial agent.

Antibiotic A/16686 is a glycopeptide antibiotic with a basic characterwhich is capable of forming acid addition salts. Therefore thephysiologically-acceptable acid addition salts of antibiotic A/16686 arepart of this invention.

For simplicity in discussions of utility, the term "antibiotic A/16686"is used herein to refer to an antibiotic selected from antibioticA/16686 free base and its physiologically-acceptable acid additionsalts.

Antibiotic A/16686 inhibits in vitro the growth of certain pathogenicbacteria, especially gram-positive. Moreover parenteral administrationof antibiotic A/16686 gives a high degree of protection againstexperimental infections in mice.

As stated above, antibiotic A/16686 is produced by culturing a novelstrain of the Actinoplanes genus. A culture of this strain, which wasisolated from a soil sample collected at Vaghalbod (India), has beendeposited on Jan. 30, 1979 with the permanent culture collection of ATCC(American Type Culture Collection)-12301 Parklawn Drive, Rockville-Md.20852-U.S.A. where it has been accorded the accession number ATCC 33076.

The characteristics of Actinoplanes sp. ATCC 33076 are given in thefollowing paragraphs.

Morphology

The strain grows well on different media with a orange color of thesubstrate mycelium. It does not produce pigment. Aerial mycelium isalways absent. At microscopic examination the vegetative myceliumreveals branched hyphae with a diameter of about 1 μm. The sporangiaform scantly only on potato-agar and are globose with a very irregularsurface and a diameter ranging from 5.0 to 9.0 μm. Sporangial release isobserved after rupture of the wall of the sporangium. The subsphericalspores are motile (1.0-1.5 μm diameter). Analysis of the cell-wallcomponents reveals meso-diaminopimelic acid and sugar pattern of type D(Lechevalier et al.,--Chemical composition as a criterium in theclassification of Actinomycetes. Adv. Applied Microbiology, 14, 1971.Academic Press N.Y.)

Cultural characteristics

Table 1 reports the cultural characteristics of Actinoplanes ATCC 33076cultivated on various standard media suggested by Shirling and Gottlieb(Intern. J. System. Bact. 16, 313-340, 1966) and other media recommendedby Waksman (The Actinomycetes, Vol. II-The Williams and Wilkins Co.1961). The cultural characteristics were determined after 6 to 14 daysof incubation at 30° C.

                  TABLE I                                                         ______________________________________                                        Cultural characteristics                                                      The number of some of the culture media refers to those                       given by Shirling and Gottlieb in Methods for characte-                       rization of Streptomyces species - Intern. J. System.                         Bact. 16, 313-340, 1966.                                                      Culture media     Cultural characteristics                                    ______________________________________                                        Medium No. 2 (yeast extract-                                                                    Abundant growth, wrinkled                                   malt agar)        surface, light brown 12 H 12                                Medium No. 3 (oatmeal agar)                                                                     Scant growth, thin, light                                                     orange 9 B 6                                                Medium No. 4 (inorganic salts-                                                                  Moderate growth, crusty sur-                                starch agar)      face, orange 11 L 12                                        Medium No. 5 (glycerol-aspara-                                                                  Scant growth, hyaline                                       gine agar)                                                                    Medium No. 6 (peptone-yeast                                                                     Scant growth, hyaline to                                    extract-iron agar)                                                                              light brown                                                 Medium No. 7 (tyrosine agar)                                                                    Scant growth, smooth surface,                                                 brown 5 D 11                                                Oatmeal agar (according to                                                                      Abundant growth, wrinkled                                   Waksman)          surface, orange to brown                                                      12 C 10                                                     Hickey and Tresner's agar                                                                       Abundant growth, crusty                                                       surface, orange 11 G 8                                      Czapek glucose agar                                                                             Moderate growth, crusty                                                       surface, orange 11 G 8                                      Glucose asparagine agar                                                                         Scant growth, crusty surface                                                  light orange 11 F 6                                         Nutrient agar     Moderate growth, smooth sur-                                                  face, orange 11 G 8                                         Potato agar       Abundant growth, wrinkled                                                     surface, amber-brown 12 E 10                                Bennett's agar    Abundant growth, wrinkled                                                     surface, orange 11 C 8                                      Calcium malate agar                                                                             Moderate growth, smooth sur-                                                  face, light orange 10 C 6                                   Skim milk agar    Abundant growth, wrinkled                                                     surface, orange 9 C 2                                       Czapek agar       Moderate growth, crusty sur-                                                  face, orange 10 D 7                                         Egg agar          Moderate growth, smooth sur-                                                  face, hyaline to light orange                               Peptone glucose agar                                                                            Abundant growth, wrinkled                                                     surface, orange 11 G 11                                     Agar              Very scant growth, smooth                                                     surface, hyaline                                            Loeffler serum    Very scant growth, smooth                                                     surface, orange                                             Potato            Scant growth, crusty, light                                                   brown                                                       Gelatin           Scant growth, light orange                                  Cellulose         Very scant growth, thin, hya-                                                 line                                                        ______________________________________                                    

Carbon utilization

Table II reports the utilization of carbon sources examined according tothe method of Pridham and Gottlieb (J. Bact. 56, 107, 1948).

                  TABLE II                                                        ______________________________________                                        Carbon sources  Utilization                                                   ______________________________________                                        Inositol        -                                                             Fructose        +                                                             Rhamnose        +                                                             Mannitol        -                                                             Xylose          +                                                             Raffinose       -                                                             Arabinose       +                                                             Cellulose       -                                                             Sucrose         +                                                             Glucose         +                                                             Mannose         +                                                             Lactose         -                                                             Salicin         +                                                             ______________________________________                                         + = positive utilization                                                      - = no growth                                                            

Physiological characteristics

Table III reports the physiological characteristics of the strain.

                  TABLE III                                                       ______________________________________                                        Test                   Results                                                ______________________________________                                        Hydrolysis of starch   positive                                               H.sub.2 S formation    positive                                               Tyrosinase reaction    negative                                               Casein hydrolysis      positive                                               Solubilization of calcium malate                                                                     negative                                               Liquefaction of gelatine                                                                             positive                                                ##STR1##              positive negative                                      Cellulose decomposition                                                                              negative                                               ______________________________________                                    

For producing antibiotic A/16686 the strain Actinoplanes sp. ATCC 33076is cultivated under aerobic conditions in an aqueous nutrient mediumcontaining an assimilable source of carbon, an assimilable source ofnitrogen and inorganic salts. Said culture medium can be anyone of anumber of nutrient media usually employed in the fermentation art,however certain media are preferred. Thus, for instance, preferredcarbon sources are glucose, fructose, mannose, sucrose and the like.Preferred nitrogen sources are soybean meal, peptone, meat extract,yeast extract, tryptone, amino acids and the like. Among the inorganicsalts which can be incorporated in the culture media are the customarysoluble salts capable of yielding sodium, potassium, iron, zinc, cobalt,magnesium, calcium, ammonium, chloride, carbonate, sulfate, nitrate andlike ions. Ordinarily the antibiotic-producing strain is precultured ina shake flask, then the culture is used to inoculate jar fermentors forproduction of substantial quantities of antibiotic A/16686. The mediumused for the preculture can be the same as that employed for largerfermentations, but other media can also be employed.

The A/16686-producing strain can be grown at temperatures between about20' C. and about 37° C. and preferably at temperatures of about 28°-30°C.

During the fermentation, antibiotic production can be followed bytesting samples of the broth or of extracts of the mycelial solids forantibiotic activity. Organisms known to be sensitive to antibioticA/16686 are useful for this purposes. One especially useful assayorganism is Sarcina lutea. The bioassay is conveniently performed by theagar diffusion method on agar plates. Maximum production of antibioticactivity generally occurs between about the third and the fifth days.The antibiotic produced during fermentation of the strain Actinoplanessp. ATCC 33076 is found both in the broth and in the mycelial mass.Recovery of antibiotic A/16686 may therefore, be carried out byseparated extraction of broth and mycelium.

Extraction of the mycelial mass is best accomplished with methanol, butother lower alcohols and chloroform are also suitable. AntibioticA/16686 is recovered as a raw product from the extracting solvent byroutine procedure. Analogously, also the broth is extracted-preferablywith n-butanol--and a further amount of raw antibiotic A/16686 isobtained by precipitation from this solution. Purification of the rawantibiotic A/16686 is then achieved by treating the product recoveredfrom the extracting solvents with a mixture chloroform:ethanol:water4:7:2, separating the oily product which forms and pouring it in water.This treatment causes solidification of the product which is recoveredby filtration, and further purified by Silicagel column chromatographyeluting with a mixture acetonitrile: 0.01 N HCl 1:1. Antibiotic A/16686,which according to this procedure is recovered in the form of thehydrochloride, is then desalted by chromatography on a cross-linkeddextran gel column.

Each step of the above purification procedure is monitored by thin-layerchromatography using a suitable solvent system such as a basic solventsystem, for instance n-propanol:n-butanol:N-ammonium hydroxide 2:3:4(upper phase) or methanol: 10% aqueous ammonium acetate: 10% ammoniumhydroxide 10:9:1, in which any acid addition salt of antibiotic A/16686is converted into the free base. All the above steps are therefore aimedto isolate pure antibiotic A/16686 characterized by a particular R_(f)value in the particular solvent system employed.

Other purification methods may suitably be employed involvingconventional extraction and adsorption procedures. Said alternativemethods may be easily set up, step by step, by monitoring thepurification progress by thin-layer chromatography, as described above.Following the t.l.c. spot of antibiotic A/16686 with a particular R_(f)value, it will thus be apparent to any skilled technician whichoperations might suitably be carried out in order to isolate pureantibiotic A/16686.

If desired the pure antibiotic A/16686 hydrochloride, obtained accordingto the procedure set forth above, may then be converted into thecorresponding free base or into another physiologically-acceptable acidaddition salt by common procedures.

Antibiotic A/16686 is an antimicrobial agent and it is especially activeagainst gram-positive microorganisms. In particular, the in vitroactivity spectrum of antibiotic A/16686 is summarized in the followingTable IV:

                  TABLE IV                                                        ______________________________________                                                              M.I.C. (μg/ml)                                       Organisms             Antibiotic A/16686                                      ______________________________________                                        S. aureus ATCC 6538   0.16                                                    S. aureus ATCC 9144   0.16                                                    S. aureus Tour        0.31                                                    Staphylococcus 10B Ciba                                                                             0.08                                                    S. epidermidis ATCC 12228                                                                           0.08                                                    S. saprophyticus NCTC 7292                                                                          0.16                                                    M. flavus ATCC 10240  0.016                                                   S. lutea ATCC 9341    0.02                                                    S. pyogenes C 203 SKF 13400                                                                         0.01                                                    S. pneumoniae Felton UC 41                                                                          0.05                                                    S. faecalis ATCC 7080 0.31                                                    S. foecium ATCC 10541 0.08                                                    S. agalactiae ATCC 7077                                                                             0.02                                                    S. mutans ATCC 27607  0.08                                                    C. diphtheriae var.mitis ATCC 11051                                                                 0.31                                                    C. xerosis NCTC 9755  0.02                                                    B. subtilis ATCC 6633 0.062                                                   B. cereus var.mycoides ATCC 11778                                                                   0.08                                                    C. perfringens ISS 30543                                                                            0.16                                                    P. acnes ATCC 6919    0.4                                                     P. acnes ATCC 6922    0.8                                                     P. acnes ATCC 25746   0.4                                                     ______________________________________                                    

Table V reports the results of tests wherein antibiotic A/16686 testedagainst a variety of Staphylococcus aureus, Streptococcus pyogenes,Streptococcus pneumoniae and Streptococcus faecalis strains clinicallyisolated.

                  TABLE V                                                         ______________________________________                                                         M.I.C. (μg/ml)                                            Organisms        Antibiotic A/16686                                           ______________________________________                                        S. aureus 54310 L 1096                                                                         0.62                                                         S. aureus 54560/I L 1097                                                                       0.31                                                         S. aureus 54635 L 1098                                                                         0.31                                                         S. pyogenes L 33 0.04                                                         S. pyogenes L 794                                                                              0.08                                                         S. pyogenes L 800                                                                              0.04                                                         S. pyogenes L 801                                                                              0.16                                                         S. pyogenes L 802                                                                              0.08                                                         S. pyogenes L 803                                                                              0.08                                                         S. pyogenes L 804                                                                              0.62                                                         S. pyogenes L 805                                                                              0.08                                                         S. pneumoniae L 1055                                                                           0.04                                                         S. pneumoniae L 1102                                                                           0.04                                                         S. pneumoniae L 1174                                                                           0.08                                                         S. faecalis L 768                                                                              0.31                                                         S. faecalis L 876                                                                              0.31                                                         S. faecalis L 922                                                                              0.31                                                         S. faecalis L 949                                                                              0.31                                                         S. faecalis L 965                                                                              0.31                                                         S. faecalis L 1139                                                                             0.62                                                         S. foecium L 763 0.16                                                         ______________________________________                                    

Antibiotic A/16686 has also been found to possess a high order ofactivity in vivo against various pathogenic organisms. The effectivenessof antibiotic A/16686 is readly apparent from Table VI which gives theED50 values in mice against three different microorganisms.

                  TABLE VI                                                        ______________________________________                                        ED.sub.50 mg/kg s.c.                                                                           S.pyogenes  S.pneumoniae                                     S.aureus Tour    C203 ISM    Felton UC 41                                     ______________________________________                                        A/16686 24.6         0.09        0.2                                          ______________________________________                                    

Antibiotic A/16686 has been found to possees a relatively low level oftoxicity when used in test animals. For example, the LD₅₀ value, whenthe antibiotic is administered intraperitoneally to mice isapproximately comprised between 500 and 750 mg/Kg.

A further object of the present invention is therefore the use ofantibiotic A/16686 as an antibacterial agent, intending with the term"use" all industrially applicable aspects and acts of said use,including the embodying of antibiotic A/16686 into pharmaceuticalcompositions, which therefore represent a further feature of the presentinvention.

These pharmaceutical compositions, suitable for oral, topical orparenteral administrations, are prepared in the usual ways known to allskilled in the pharmaceutical sciences. Examples of these formulationsare described for instance in Remington's Pharmaceutical Sciences15^(th) Ed., 1975 Mack Publishing Co. Easton, Pa. These forms includetablets, capsules, powders, ointments, liquid solutions, solutions forinjection and the like. The dosage unit may contain from 0.5 to 99percent, preferably from 5 to 80 percent of active ingredient. The dailydosage may be set up considering several factors such as the bodyweight, the infecting organism, the severity of the infection, theperiod and the mode of administration.

In order to illustrate more fully the operation of this invention, thefollowing examples are provided.

EXAMPLE 1 Fermentation of the strain Actinoplanes sp. ATCC 33076

A culture of Actinoplanes sp. ATCC 33076 is precultured by growing thestrain in a shake-flask culture having the following composition

    ______________________________________                                        meat extract         3 g/l                                                    yeast extract        5 g/l                                                    tryptone             5 g/l                                                    soluble starch       24 g/l                                                   glucose              1 g/l                                                    CaCO.sub.3           4 g/l                                                    Tap water            1 liter                                                  ______________________________________                                    

The flasks are shaken for about 96 hours at 28°-30° C. and then thepre-cultures (1 liter) are used to inoculate the jar fermentors eachcontaining 10 liters of the following nutrient medium

    ______________________________________                                        Meat extract         40 g                                                     Peptone              40 g                                                     Yeast extract        10 g                                                     Sodium chloride      25 g                                                     Soybean meal         100 g                                                    Glucose              250 g                                                    CaCO.sub.3           50 g                                                     Tap water            10 liters                                                ______________________________________                                    

The fermentation batches are incubated aerobically under stirring at28°-30° C. At intervals, the antibiotic activity is assayedmicrobiologically by the agar diffusion method using Sarcina lutea asthe test organiism. The maximum activity is reached after 72 to 120hours of fermentation.

EXAMPLE 2 Recovery of antibiotic A/16686

Whole fermentation broth (170 l) prepared as described in Example 1 iscooled at 10° C. and brought to pH 3.5 by means of 18% HCl. Theresulting acidic broth is filtered using a filter aid (Clarcel Flow-Ma),and the mycelial cake is washed with water. Then the filtered broth andthe mycelium are further processed separately.

(a) Methanol (30 l) is used to extract the mycelial mass which, afterfiltration, is extracted again with a mixture methanol/water (30 l ofmethanol plus 5 l of water). The exhausted mycelium is discarded and thetwo methanol extracts are concentrated under vacuum at a temperaturelower than 40° C. to yield an aqueous concentrate (6 l). This aqueousconcentrate is extracted with three portions, 10 l each, of n-butanol,which are combined and concentrated to a small volume under vacuum.

This concentrate is added to petroleum ether and the resultingprecipitate is separated by decantation and added to a further amount ofpetroleum ether. The precipitate is separated by filtration and driedunder vacuum at room temperature to give 80 g of antibiotic A/16686 as araw material having a M.I.C. against S. pneumoniae UC 41 of 0.1 μg/ml.

(b) The filtered broth plus washing (165 l) is cooled to 10° C. andadjusted to pH 3.5 by addition of concentrated HCl (2.5 l). The obtainedsolution is extracted with n-butanol (80 l) and the organic extract isthen concentrated under vacuum at a temperature lower than 35° C., togive a butanol concentrate (6 l). This concentrate is added to petroleumether and the obtained precipitate, separated by decantation is added toa further amount of petroleum ether. The solid is separated byfiltration and dried under vacuum at room temperature to give 26.3 g ofraw antibiotic A/16686 having a M.I.C. against S. pneumoniae UC 41 of0.8 μg/ml.

EXAMPLE 3 Purification of antibiotic A/16686

47.7 g of the raw antibiotic A/16686 obtained in Example 2a) are treatedwith 1.4 l of a chloroform: ethanol:water mixture (4:7:2) (v/v/v) andthe oily product which forms is separated from the solution bydecantion. Further 70 ml of the above mixture are then added to the oilyproduct and the separation is repeated. By treatment of the oily productwith water (440 ml), it solidifies and is separated by centrifugationand filtration:

(a) The solid, which is separated, is suspended in water (170 ml),dissolved by addition of methanol (400 ml) and filtered. Then thesolvents are stripped under vacuum by adding n-butanol at a temperaturenever higher than 35° C., to give a butanol concentrate of about 50 ml.By addition of diethyl ether (500 ml) a precipitate forms which isseparated by filtration and dried under vacuum at room temperatureyielding 1.012 g of rather pure antibiotic A/16686 having a M.I.C. on S.pyogenes of 0.025 μg/ml. The rather pure antibiotic A/16686 so obtainedis then submitted to the following purification procedures:

1.58 g of the above antibiotic A.16686 characterized by a M.I.C. againstS. pyogenes of 0.025 μg/ml are dissolved in acetonitrile:water 1:1 (v/v)and the resulting solution is applied to a column containing 430 g ofsilica gel 60 (Merck 0.06-0.2 mm) prepared in the same mixture. Thecolumn is developed first using the same acetonitrile-water mixture andcollecting 70 fractions of 20 ml each, and then using acetonitrile:N/100 HCl 1:1 (v/v) and collecting further 290 fractions, of 20 ml each.Elution of the column is monitored by thin layer chromatography on 60F₂₅₄ silica gel plates and by assaying fractions against Sarcina lutea.Fractions 130 to 265 are combined and the solvents are stripped undervacuum with n-butanol to give a n-butanol concentrate of 20 ml. Thisresidual volume is poured into a large amount of ethyl ether and theprecipitate which forms is separated by filtration and dried undervacuum at room temperature over P₂ O₅ yielding 1.015 g of antibioticA/16686.

0.67 g of the above substance are dissolved in 24 ml of water and 76 mlof methanol. The resulting solution is applied to a 3.0×62.0 cm columncontaining 220 g of Sephadex LH-20, prepared in methanol:water 7:3(v/v). The column is developed with the same mixture collecting 10 mlfractions. Fractions 24 to 32 are combined and the solvents are strippedunder vacuum at a temperature lower than 35° C. with n-butanol to aresidual butanol volume of about 10 ml. This solution is added to ethylether to precipitate the desired pure antibiotic A/16686. Theprecipitate is separated by filtration washed with ethyl ether and driedunder vacuum over P₂ O₅ at room temperature yielding 0.26 g of pureantibiotic A/16686.

(b) The filtrate is stripped under vacuum at a temperature lower than35° C. by adding n-butanol, to give a n-butanol concentrate of 50 ml. Byaddition of petroleum ether a precipitate forms which is separated byfiltration and dried under vacuum at room temperature yielding 42.9 g ofpartially purified antibiotic A/16686 characterized by a M.I.C. valueagainst S. pyogenes of 0.2 μg/ml. This antibiotic substance is suspendedin water (2.5 l) and dissolved by addition of NaOH 1 N up to pH 7. Thenthe obtained aqueous solution is extracted with three portions, 5 leach, of n-butanol and this organic phase, after being washed withwater, is concentrated to 200 ml under vacuum at a temperature lowerthan 35° C.

By adding diethyl ether to the concentrate, a precipitate forms which isfiltered and dried under vacuum at room temperature. The dried productis dissolved in 160 ml of the upper layer of a n-propanol:n-butanol-1 Nammonium hydroxyde mixture 2:3:4 (v/v/v). The resulting solution isapplied to a 100 cm high column, 7.5 cm in diameter, containing 1.7 Kgof silica gel 60 (Merck 0.06-0.2 mm) prepared in the above solventsmixture. The column is developed using the same mixture collecting 300ml fractions. Elution of the column is monitored by thin layerchromatography. Fractions 21 to 26 are combined and stripped undervacuum at a temperature lower than 35° C. with n-butanol to give an-butanol concentrate of 50 ml. By addition of diethyl ether aprecipitate forms which is separated by filtration, washed with diethylether and dried under vacuum over P₂ O₅ at room temperature. A 1.875 gcrop of rather pure antibiotic A/16686 is obtained which is then furtherpurified by following the same procedures described under a) above.

Antibiotic A/16686 as the hydrochloride, is a white, crystallinematerial slightly hydroscopic which melts at 224°-226° C. It is verysoluble in water and dimethylformamide, soluble in methanol, ethanol,propanol and butanol, but is insoluble in ethyl ether, petroleum etherand benzene. Elemental analysis of antibiotic A/16686 hydrochloride,previously dried at 140° C. under inert atmosphere, indicates thefollowing approximate percentage composition (as an average of severalanalyses): carbon 51.73%; hydrogen 6.34%; nitrogen 9.96%; chlorine(total content) 5.84%; chlorine ions 4.74%; and residue 1%. The infraredabsorption spectrum was determined in nujol.

The following absorption maxima are observed (in cm⁻¹): 3290-3070, 2930and 2860 (nujol), 1765, 1630, 1510, 1455, 1375 (nujol), 1230, 1175,1130, 1065, 1030, 1015, 980, 840 and 820.

The ultraviolet absorption spectrum exhibits the following absorptionmaxima:

    ______________________________________                                        (a) in methanol                                                                      232 nm        (E.sup.1% .sub.1cm = 178)                                       265 nm        (E.sup.1% .sub.1cm = 107)                                (b) in methanol containing 0.1N HCl                                                  231 nm        (E.sup.1% .sub.1cm = 167)                                       270 nm        (E.sup.1% .sub.1cm = 96)                                 (c) in methanol containing 0.1N NaOH                                                 250 nm        (E.sup.1% .sub.1cm = 232)                                       295 nm        (shoulder)                                               (d) in methanol containing pH 7.38 buffer                                            231 nm        (E.sup.1% .sub.1cm = 167)                                       270 nm        (E.sup.1% .sub.1cm = 96)                                 ______________________________________                                    

The ultraviolet absorption spectra were registered with a Beckmann DK-2Spectrophotometer). Antibiotic A/16686 hydrochloride has a specificrotation, [α]_(D) ²⁴, of +49.7° (c=0.43% in DMF).

Antibiotic A/16686 shows the following characteristic reaction:

    ______________________________________                                        ninhydrin (3% ethanolic solution)                                                                    negative                                               ninhydrin (3% ethanolic solution                                                                     positive                                               + sodium acetate)      positive                                               1% FeCl.sub.3 - 1% K.sub.3 Fe(CN).sub.6 (aqueous                                                     positive (green)                                       solution)                                                                     Molisch                positive                                               Fehling                negative                                               Biuret                 positive                                               Anthrone               positive                                               Millon                 negative                                               H.sub.2 SO.sub.4 conc  negative                                               KMnO.sub.4 aqueous     positive                                               ______________________________________                                    

The R_(f) values of antibiotic A/16686 in paper chromatography usingdifferent elution systems and S. aureus ATCC 6538 as a detectionorganism, are given in the following Table:

                  TABLE VII                                                       ______________________________________                                        Chromatographic behaviour (Whatman Nr. 1 paper)*                              of antibiotic A/16686                                                         Elution systems           R.sub.f values                                      ______________________________________                                        (1)   n-butanol saturated with Sorensen                                                                     0.00                                                  buffer pH 6.0                                                           (2)   n-butanol saturated with water                                                                        0.00                                                  containing 2% of p-toluenesulfonic                                            acid                                                                    (3)   n-butanol saturated with water containing                                                             0.00                                                  2% ammonium hydroxide                                                   (4)   Sorensen buffer, pH 6.0, saturated                                                                    0.05                                                  with n-butanol                                                          (5)   n-butanol:methanol:water 4:1:2                                                                        0.43                                            (6)   ethyl acetate saturated with water                                                                    0.00                                            (7)   n-butanol:acetic acid:water 2:1:1                                                                     0.52                                            (8)   n-butanol:pyridine:water 4:3:7                                                                        0.87                                            ______________________________________                                         *Descending chromatography                                               

Rm: 40 cm

Amounts: 20 μg of the compound dissolved in water methanol (2 mg/ml)

The R_(f) values of antibiotic A/16686 in various thinlayerchromatography systems are listed in the following table (the conditionsare indicated below the table):

                  TABLE VIII                                                      ______________________________________                                        Elution systems (v/v/v)     R.sub.f values                                    ______________________________________                                        (1)  n-propanol:n-butanol:N ammonium                                                                          0.15                                               hydroxide 2:3:4 (upper phase)                                            (2)  n-butanol:acetic acid:water 4:2:5                                                                        0.61                                          (3)  n-butanol:ethanol:0.1 N hydrochloric                                                                     0.61                                               acid                                                                     (4)  chloroform:ethanol:10% acetic acid 4:7:2                                                                 0.00                                          (5)  n-butanol:acetic acid:water 4:1:5                                                                        0.17                                          (6)  methanol:10% aqueous ammonium acetate:10%                                                                0.52                                               ammonium hydroxide 10:9:1                                                (7)  n-butanol:pyridine:water:acetic acid                                                                     0.45                                               6:4:3:1                                                                  (8)  methanol:10% aqueous ammonium acetate:10%                                                                0.11                                               ammonium hydroxide 10:9:1                                                (9)  0.25 M aqueous NaH.sub.2 PO.sub.4 :acetonitrile 1:1                      (10) methanol:10% aqueous ammonium acetate 1:1                                                                0.65                                          (11) n-butanol:acetic acid:water 4:2:5                                                                        0.77                                          ______________________________________                                    

Rm: about 140 mm

Amounts: 2÷5 μl of a solution (1 mg/ml) of the compound inacetonitrile-water 1:1

Visualization:

(a) bioautography on agar plates seeds with B. subtilis ATCC 6633; (b)carbonization by heating with α-naphthol-sulfuric acid; (c) Jodinevapour; (d) chlorine-toluidine reagent; (e) UV-light at 254 nm

1 to 7-Silicagel 60 F₂₅₄ plates (Merck); Visualization a, e, b, c, d, e

8,9-Silicagel 60 F₂₅₄ silanised (Merck); Visualization e 10,11-Cellulose F plates (Merck); Visualization a,

As determined through high performance chromatographic techniques,antibiotic A/16686, isolated and purified as described in Example 3,consists of a main component which amounts to 70-80%, while theremaining 30-20 % is ascribable to al least two minor components.Antibiotic A/16686 hydrochloride isolated as described in the preceedingexamples is the hydrochloride of a chlorine-containing basicglycopeptide which differs from antibiotics belonging to the classicalglycopeptide group in containing chlorine and in the variety of itsamino-acids components. In fact amino-acid analysis of antibioticA/16686, after acid hydrolysis is 6 N hydrochloric acid at 110° C. for 6hours, by means of an amino-acid authoanalyzer, indicated the presenceof the following amino-acids: ornithine (118 μg/mg=0.58 μM/mg), asparticacid (29.2 μg/mg=0.22 μM/mg), threonine (68 μg/mg=0.57 μM/mg), glycine(25 μg/mg=0.33 μM/mg), alanine (29 μg/mg=0.32 μM/mg), leucine (41μg/mg=0.31 μ M/mg) and phenylalanine (42 μg/mg=0.25 μM/mg).

Further four peaks were detected using the column for neutral and acidicamino acids. Two of these four amino-acids were identified by means ofgas chromatography-mass spectrometry after conversion into theircorresponding methyl ester-trifluoroacetyl derivatives and were assignedthe following structures: ##STR2##

EXAMPLE 4 Isolation of the free base

By treatment of a solution of antibiotic A/16686 hydrochloride (0.05 g)in water (3 ml)--ethanol (8 ml) with 1,2-epoxybutane (2 ml) aprecipitate forms which after standing at low temperature for one day isseparated by centrifugation, washed with ethanol and dried over P₂ O₅under vacuum at room temperature yielding 0.016 of the correspondingfree base.

Treatment of said free basic form of antibiotic A/16686 with apharmaceutically acceptable inorganic or organic acid leads to theformation of a corresponding acid addition salt. Pharmaceuticallyacceptable acids are non-toxic acids that are suitable for the formationof therapeutically useful salts as known in the art such as for instancehydrochloric, hydrobromic, sulfuric, phosphoric, nitric, tartaric,acetic, succinic, lactic, glutamic or methanesulphonic acid.

We claim:
 1. A biologically pure culture of Actinoplanes sp. ATCC 33076and a nutrient medium consisting of an assimilable source of each ofcarbon, nitrogen, and essential mineral salts, said culture beingcapable of producing antibiotic A/16686 in a recoverable quantity uponfermentation.
 2. A biologically pure culture of Actinoplanes sp. ATCC33076, capable of producing antibiotic A/16686.